3. Molecular studies of tidal rhythms
The work described above suggests that tidal and circadian rhythms in foraging shorebirds reflect adjustments to the complex temporal environment in which they live. However, other factors beyond circadian day–night or tidal rhythms, such as predation or behaviour of conspecifics (which themselves may have clock-like features), may outweigh the entrainment of behaviour imposed by these geophysical variables [14]. Still, circadian rhythms are identified in nearly all higher organisms and, for example, migratory birds use the clock for navigation and to compensate for the movement of the sun [31]. Consequently, given the ubiquity of biological rhythmicity, considerable effort has been expended over five decades to identify the genetic and molecular bases for these behavioural rhythms. The discovery of the molecular basis of the circadian clock was a defining moment in the study of gene regulation of complex phenotypes [32].
Despite insects and crustaceans having long been studied for lunar-related rhythms at the behavioural level [6], we have been missing a genetically tractable model species from intertidal habitats. Here, we introduce four organisms (figure 4) where molecular interventions were recently used to illuminate the molecular bases of lunar-related rhythms. Specifically, we highlight the finding of tidal activity rhythms in the marine isopod Eurydice pulchra and the mangrove cricket, Apteronemobius asahinai, semi-lunar emergence rhythms of the marine midge, Clunio marinus, and the lunar reproductive cycles of the bristle worm Platynereis dumerilii.

Figure 4. Examples of lunar-related rhythms of invertebrates. (a) Eurydice adult with chromatophores (black dots on the dorsal surface of cuticle) and swimming activity of a single individual over 9 days in constant darkness). The animal was taken during a spring tide from Bangor, Wales, UK and placed immediately in constant darkness (DD). The approximate natural light (grey)/dark (black) cycle on the day the animal was harvested is shown as a bar above the actogram and each day’s activity is double plotted on a horizontal 48 h scale so that so that each row represents two consecutive days. Note that the movement to the right on every successive day reveals a tidal period longer than 12 h and the night-time activity is greater than that of the daytime (diurnal inequality). Adapted from [30]. (b) Mangrove cricket and an actogram for single individual placed in 12 L: 12 D for 8 days then allowed to free run in DD, during which there is more intense activity in the dark phase compared with the light phase (see the histogram) which drifts towards the right reflecting the predominantly 24.8–25.5 h rhythm which is about twice the tidal period of approximately 12.4 h. The histogram shows the night-time burst of activity (filled columns) being greater than the daytime burst (unfilled columns) for a few cycles but as this is modulated by the circadian clock, it drifts out of phase with the tidal cycle; so after many cycles, the daytime tidal episode is greater than the night-time (adapted from [33]). The cricket image is taken from http://mangrove.nus.edu.sg/guidebooks/text/2010.htm. (c) Midge C. marinus and five natural populations (i) with different phases of emergence (ii) and semi-lunar or lunar frequency during day of emergence (iii). Image is taken from https://www.flickr.com/photos/davidh-j/6270311922 and figure was adapted from [34]. (d) Premature adult, and adult male and female Platynereis dumerilii.Lunar maturation cycle of single individual over several months. FM, full moon simulated by dim light. NM, new moon. Lunar month in days plotted as horizontal yellow bar. Adapted from [35].

(a) Circadian and circatidal rhythms in a marine isopod and a mangrove cricket
Eurydice pulchra is a marine isopod that lives in the intertidal zone around northern European coasts (figure 4a). As the tide comes in, Eurydice swims out of its sandy burrow and forages. As the tide goes out, Eurydice buries itself back into the sand so it is not dragged out to sea [33,36]. In constant darkness, Eurydice exhibits an endogenous circatidal swimming rhythm of 12.4 h (figure 4a) which can be reset by vibration stimuli, and is temperature compensated, thereby showing all the hallmarks of a true clock [36]. Interestingly the swimming pattern usually shows the diurnal inequality phenomenon at temperate latitudes (figure 1b), so nocturnal high-tide swimming is considerable greater than daytime swimming (figure 4a). This modulation in swimming is regulated by the circadian clock because under bright light it is disrupted, whereas the tidal 12.4 h swimming period is unaffected, suggesting an independence of circadian and tidal oscillators [36]. Moreover, Eurydice is called the ‘speckled sea louse’ because it carries pigmented spots, chromatophores that expand during the day and contract at night (figure 4a) [33,36]. This 24 h cycle is likely regulated by a circadian clock because the 24 h cycle persists under constant darkness, can be reset by light and is disrupted by constant bright light [33,36]). Indeed, knockdown of Eurydice’s period gene, whose Drosophila orthologue plays a central role in the molecular clock machinery of Drosophila melanogaster, has a similar effect to constant light, with circadian cycles in chromatophore dispersion and in Eurydice timeless mRNA disrupted. Yet the very same canonical clock gene misregulation has little effect on the circatidal swimming periodicity of 12.4 h [36].

Although these results invoke separate circatidal and circadian oscillators, pharmacological inhibitors of Eurydice’s casein kinase 1ɛ (CK1ɛ), which phosphorylates PER protein in D. melanogaster and hence could also inhibit similar post-translational modification of Eurydice’s PER protein, lengthened both tidal swimming and the circadian chromatophore cycle [36]. This might suggest that the two oscillators share a common pathway. However CK1ɛ has many targets, so the inhibitor might render CK1ɛ less able to phosphorylate a tidally relevant protein that we have yet to identify. It is unlikely that any effect of the inhibitor on Eurydice’s PER protein phosphorylation is mediating tidal lengthening because direct disruption of Eurydice’s period gene mRNA through RNA interference had no effect on this phenotype [36].

The circadian day–night modulation of the tidal swimming rhythms in Eurydice is also observed in the locomotor activity of the mangrove cricket [37] (figure 4b). However, the periodicity of the cricket’s locomotor activity pattern is circatidal and approximately 12.4 h. Elegant genetic studies have used RNAi-mediated knockdown of the canonical clock genes in this species, period and Clock (in insects and mammals CLOCK protein is one of a pair of molecules that activate periodand timeless gene transcription). The knock-down left 12.4 h tidal rhythms intact, but disrupted the circadian modulation of alternate bouts of locomotor activity [38,39]. As in Eurydice, these gene knockdowns suggest that the two molecular oscillators underlying circadian and tidal rhythms are largely independent of each other. Moreover, surgical ablation of the optic lobes (likely location of the circadian oscillator) disrupted the circadian locomotor pattern, but as with the gene knockdown, the tidal rhythm remained intact [34]). Consequently, molecular mechanisms of the two oscillators not only may be independent, but also may reside in different groups of neurons.

(b) Circadian and semi-lunar emergence of the marine midge
Perhaps the best-known example of a moon-related phenotype in insects is the semi-lunar emergence rhythms in the marine midge, C. marinus (figure 4c), first studied by Neumann and collaborators 50 years ago (e.g. [40]). During full and new moon, millions of males and females of the midge emerge from the sea as low tide exposes the habitats where they have developed from eggs to pupae (figure 4c). These adults mate and live for a few hours, so it is critical that they emerge synchronously during those few hours of low tide. The timing of the lowest tide can be predicted from the lunar calendar, but these critical few hours during the day vary from location to location [40]). Thus, the emergence of the marine midge has to rely on two clocks, one circa-semi-lunar or circalunar, and the other circadian.

A recent and spectacular molecular genetic study used populations of midges living in different European locations (figure 4c), in combination with the fully referenced draft genome of the midge generated de novo [7], to identify the genetic bases of semi-lunar or lunar and circadian rhythms. First, the local circadian adaptations mapped to the gene encoding calcium/calmodulin-dependent kinase II.1 (CaMKII) [7]. Importantly, mutations in the homologous gene can disrupt circadian timing in the mouse [41] and D. melanogaster [35,42]. Secondly and more importantly for lunar-related phenotypes, the genetic mapping experiment localized a chromosomal region responsible for the population differences in semi-lunar versus lunar emergence timing [7]. Lack of canonical clock circadian genes mapping to this region implies that a novel timing gene (or genes) contributes to the lunar phenotype.

(c) Circadian activity and lunar reproductive cycles of the bristle worm
Finally, the bristle worm P. dumerilii (figure 4d) spawns in a monthly rhythm, in which the number of worms that are sexually mature peaks around the time of new moon and troughs at full moon (figure 4d) [43,44]. This monthly rhythm appears to be driven by exposure to moonlight during full moon because the monthly cycle of reproductive maturity can be entrained in the laboratory by nocturnal dim light lasting for eight consecutive nights during the month (figure 4d). Also, the monthly maturity rhythm will free-run for several months under constant darkness, but not under constant light or in constant darkness without previous moonlight exposure, suggesting a true circalunar cycle [43]. In addition, the worms show circadian locomotor rhythms particularly in light–dark cycles. The strength of this rhythm is modulated by the phases of the moon, suggesting a crosstalk between the two oscillators [43].
When the worms were treated with the same CK1ɛ/δ kinase inhibitor used in Eurydice, circadian locomotor behaviour and circadian gene expression of canonical clock genes were severely disrupted, but the circalunar maturity rhythm was essentially unaffected. The authors’ conclusions resonated with those from Eurydice and the mangrove cricket, in that the circadian oscillators appeared to be molecularly independent from the circalunar clocks [43]. The only possible inconsistency between the discussed studies concerns tidal and lunar periodicity. The CK1ɛ inhibitor influenced the tidal periodicity in Eurydice, but not the lunar cycle in bristle worm. Likely, there are important differences in the mechanisms that generate 12.4 h tidal and 29 day lunar rhythms even though they are clearly geophysically and astronomically related. However, the maturity rhythm of the bristle worm was monitored only for two months after the inhibition. Thus, a period difference between the inhibited and control animals might have gone undetected. It would require several more months of expensive drug exposure and several cycles of monitoring of the maturity rhythm to state definitively that there was no effect on the period of the free-running maturation cycle.
The above examples used molecular manipulations in vivo allied to the analysis of behavioural and molecular phenotypes in non-model invertebrates. Such analyses are much more difficult to perform compared with model organisms like D. melanogaster or the mouse but they have led to an understanding of what does NOT constitute the tidal oscillator. From three independent studies in Eurydice, mangrove crickets and the bristle worm, the consensus of opinion suggests that lunar-related rhythms may not be generated by the canonical circadian clock genes. Some caution should still be reserved in accepting this conclusion, particularly concerning the CK1ε inhibitor, which dramatically affects the period of Eurydice’s tidal swimming. In addition, if the tidal oscillator in the mangrove cricket is more robust than the circadian oscillator that modulates its tidal locomotor episodes, then RNAi-mediated knockdown may not knock-down period or Clock genes far enough to affect the tidal oscillator. Unfortunately, both organisms are difficult to rear in the laboratory so the use of gene editing tools to create null-mutants is unlikely in the near future.

4. General conclusion and outlook
We have documented the crosstalk between the tidal and circadian rhythms in the distance that a red knot moved from its roost during foraging (figure 3). This is reminiscent of the circadian modulation of tidal behaviour observed in both Eurydice and the mangrove cricket. Thus, we suspect that in all these organisms the brain centres dedicated to expressing tidal and circadian phenotypes will be anatomically connected and, therefore, signalling reciprocally to each other.
The next challenge is to find which genes encode tidal/lunar time in the above-described invertebrates. Once invertebrate lunar/tidal genes are identified, homology should allow the isolation of similar genes in vertebrates like red knots. We might predict that the tidal genes that generate the approximately 12.4 h behavioural cycles might also encode cycling mRNAs by analogy with their circadian counterparts. Might these (as yet unidentified) putatively 12 h tidally cycling mRNAs show among-individual fluctuations to account for the variation in tidal rhythms observed in red knots? Could these mRNAs still be cycling in the biparental incubating species but their output is suppressed? Would any future identification of a tidally cycling mRNA in a tidal vertebrate suggest a co-option of a previously 12 h cycling mRNA in a terrestrial circadian species [45,46] that was re-used to generate tidal phenotypes when the species moved to an intertidal environment?
Whatever the identity of these tidal or lunar genes, the conservation of circadian genes in invertebrates and vertebrates might suggest that the same will be true also for tidal and lunar genes [47]. Tidal genes will initially be identified in invertebrates, but homology with vertebrate genes will be expected to open up interesting possibilities for mechanistic studies of the clock in intertidal birds. For example, using in situ hybridization will identify the brain regions that have tidally cycling molecules and comparing these regions with those areas that show circadian cycling molecules will detect both oscillators.
In addition, we must not forget the obvious, that behavioural ecology scenarios are far more complex than those we play out in the confines of the laboratory. As we have learned with shorebirds, individuals vary in their foraging rhythms, and behavioural rhythms during incubation are very loosely coupled to the major environmental cycles [14]. Consequently, the modulation of molecular rhythms by other selection pressures will provide a novel background against which to study biological rhythmicity within an ecologically realistic framework. Indeed, when rodents or flies are placed in semi-natural environments and their circadian rhythms monitored, quite startling results can be observed that could not have been predicted from laboratory studies and which question some of the assumptions made about the adaptive value of the circadian clock [48–50, but see also 51]. As with the incubation study of biparental shorebirds [14], when realistic scenarios are used to study biological rhythms, the results do not meet expectations. We, therefore, encourage behavioural ecologists and chronobiologists to seek collaborations, particularly as the long-term spatial and temporal monitoring of individuals in the field becomes feasible [52] and the new post-genomic age allows molecular study of organisms other than laboratory flies or mice. We anticipate that a fertile hybrid area of research will evolve, perhaps slowly at first, but with a real potential to significantly illuminate our understanding of the functional and adaptive roles of biological rhythms.